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We compared killing activity of micafungin in time-kill experiments in RPMI-1640 with and without 50% serum against Candida albicans, Candida dubliniensis and Candida africana reference strains and clinical isolates. Killing rates (k values) were determined for each strain and concentration. In RPMI-1640 MIC ranges were 0.015-0.03, 0.015-0.03 and 0.015 mg/L against C. albicans, C. dubliniensis and C. africana, respectively. In 50% serum MIC values for the three species increased 16- to 64-fold. In RPMI-1640 micafungin was fungicidal against two of three C. albicans isolates at 16 and 32 mg/L within 14.54 h and fungistatic against all C. africana and C. dubliniensis. Fifty per cent serum significantly decreased the growth rate of C. africana, but not of the other two species; weak in vivo replication ability of C. africana was confirmed in murine model. In 50% serum micafungin at 0.25 and 1 mg/L did not inhibit any of the three species (k values were always negative). Micafungin killing rate in 50% serum at 4, 16 and 32 mg/L was significantly decreased for C. albicans, but increased for C. dubliniensis compared to RPMI-1640. Killing activity of micafungin against C. africana was comparable or higher in 50% serum than in RPMI-1640. Although micafungin is a highly protein-bound drug, it was equally effective against the species of the C. albicans complex in 50% serum at therapeutic trough concentration (4 mg/L). Both in vitro and in vivo data confirmed the low virulence of C. africana compared to the two sibling species.

We compared the micafungin killing rate and postantifungal effect (PAFE) at 4, 16 and 32 mg/L in RPMI- 1640 and in 50% serum against the C. albicans complex. In RPMI-1640 PAFEs were 1.5 - >19.4, 9.7 - >20.1 and 15.9 - >18.5 hours for C. albicans, C. africana and C. dubliniensis, respectively. In 50% serum PAFEs decreased sharply to 0-1.7 hours for all three species; killing rates were always negative. Short growth inhibition without killing in 50% serum suggests that micafungin PAFE has a limited role in the eradication of the C. albicans complex from the bloodstream.

Currently, echinocandins are first-line drugs for treatment of invasive candidiasis. However, data on how serum influences killing activity of echinocandins against uncommon Candida species are limited. Therefore, the killing activity of micafungin in RPMI-1640 and in 50% serum was compared against Candida guilliermondii, Candida lusitaniae, and Candida kefyr. Minimum inhibitory concentration (MIC) ranges in RPMI-1640 were 0.5-1, 0.12-0.25, and 0.06-0.12 mg/L, respectively. In 50% serum, MICs increased 32- to 256-fold. In RPMI-1640 ≥ 0.25, ≥4, and 32 mg/L micafungin was fungicidal against all four C. kefyr (≤4.04 hours), two of three C. lusitaniae (≤16.10 hours), and two of three C. guilliermondii (≤12.30 hours), respectively. In 50% serum, all three species grew at ≤4 mg/L. Micafungin at 16-32 mg/L was fungicidal against all C. kefyr isolates (≤3.03 hours) and at 32 mg/L was fungistatic against one of three C. lusitaniae isolates. Two C. lusitaniae isolates and all three C. guilliermondii grew at all tested concentrations. Adding human serum to susceptibility test media drew attention to loss of fungicidal or fungistatic activity of micafungin in the presence of serum proteins, which is not predicted by MICs in case of C. kefyr and C. lusitaniae in RPMI-1640. Our results strongly suggest that micafungin and probably other echinocandins should be used with caution against rare Candida species.

The in vitro activities of caspofungin and micafungin were determined with and without farnesol against Candida parapsilosis biofilms. Drug interactions were examined using the XTT colorimetric assay-based broth microdilution chequerboard method. Drug-drug interactions were assessed utilising the FICI, Bliss independence models and time-kill experiments. Median sessile MICs of five C. parapsilosis clinical isolates ranged between 32-256 mg/L, 16-512 mg/L and >300 μM for caspofungin, micafungin and farnesol, respectively. Median MICs for caspofungin and micafungin in combination with farnesol showed 8-64- and 4-64-fold decreases, respectively. Paradoxical growth noticed with both echinocandins was eliminated by farnesol. Based on FICIs for sessile clinical isolates, synergism was observed for caspofungin (range of median FICIs, 0.155-0.5) and micafungin (range of median FICIs, 0.093-0.5). Concordantly, MacSynergy analysis and global fitting of non-linear regression based on a Bliss independence models also showed synergism for caspofungin and micafungin. In line with FICI findings and the Bliss independence model, synergistic interactions were confirmed by time-kill experiments. The metabolic activity of fungal cells was significantly inhibited by caspofungin+farnesol at all three tested combinations (4 mg/L+75 μM, 8 mg/L+75 μM and 16 mg/L+75 μM) between 3 and 24 h compared with the control (P<0.05-0.001). Significant inhibition was observed for micafungin+farnesol between 3 and 12h (P<0.001) but not at 24 h. Despite the favourable effect of farnesol in combination with echinocandins, further in vivo studies are needed to confirm its therapeutic advantage in catheter-associated infections caused by C. parapsilosis., (Copyright © 2016 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Previous studies suggested that caspofungin dose escalation against Candida species is more beneficial than currently used lower daily doses. Thus, we determined in vitro and in vivo activity of caspofungin against six wild-type C. albicans clinical isolates, the ATCC 10231 strain and an echinocandin resistant strain. MIC ranges of clinical isolates in RPMI-1640 with and without 50% serum were 0.125-0.25 and 0.015-0.06 mg/L, respectively. Two and three isolates showed paradoxical growth in MIC and time-kill tests, respectively, in RPMI-1640 but not in 50% serum. Caspofungin killing rate (k) in RPMI-1640 at 1 mg/L was higher than at 16 and 32 mg/L for all isolates (p<0.001). Killing rates for five of six isolates were concentration independent between 1-32 mg/L in 50% serum (p>0.05 for all comparisons), but for one isolate k value at 32 mg/L was significantly lower than at 1-16 mg/L. Although k values at 1-32 mg/L showed a great variability in 50% serum (the lowest and highest k value ranges were 0.085-0.109 and 0.882-0.985 1/h, respectively), daily 3, 5 and 15 mg/kg caspofungin was effective in a neutropenic murine model against all isolates, without significant differences between the effective doses. This study confirms that paradoxical growth does not affect the in vivo efficacy of caspofungin. We demonstrated that dose escalation did not increase the efficacy of caspofungin against C. albicans either in vitro or in vivo. These results are in concordance with the clinical experience that efficacy of echinocandins does not increase at larger doses.

Echinocandins are recommended as first-line agents against invasive fungal infections caused by Candida glabrata, which still carry a high mortality rate. Dose escalation of echinocandins has been suggested to improve the clinical outcome against C. glabrata. To address this possibility, we performed in vitro and in vivo experiments with caspofungin against four WT C. glabrata clinical isolates, a drug-susceptible ATCC 90030 reference strain and two echinocandin-resistant strains with known FKS mutations. MIC values for the clinical isolates in RPMI 1640 were ≤ 0.03 mg l(-1 ) but increased to 0.125-0.25 mg l(-1 )in RPMI 1640+50% serum. In RPMI 1640+50% serum, the replication of C. glabrata was weaker than in RPMI 1640.Caspofungin in RPMI 1640 at 1 and 4 mg l(-1) showed a fungicidal effect within 7 h against three of the four clinical isolates but was only fungistatic at 16 and 32 mg l(-1) (paradoxically decreased killing activity). In RPMI 1640+50% serum, caspofungin at ≥ 1 mg l(-1) was rapidly fungicidal (within 3.31 h) against three of the four isolates. In a profoundly neutropenic murine model, all caspofungin doses (1, 2, 3, 5 and 20 mg kg(-1) daily) decreased the fungal tissue burdens significantly (P < 0.05-0.001) without statistical differences between doses, but the mean fungal tissue burdens never fell below 105 cells (g tissue)(-1). The echinocandin-resistant strains were highly virulent in animal models and all doses were ineffective. These results confirm the clinical experience that caspofungin dose escalation does not improve efficacy.

It was previously demonstrated that brief (≤1 h) exposures to echinocandins are as effective to kill Candida albicans cells as continuous 24-h exposure. However, killing rates after continuous and short (1 h) echinocandin exposures to C. albicans have not yet been evaluated in RPMI-1640 with and without 50 % serum. We evaluated four echinocandin susceptible C. albicans bloodstream isolates, ATCC 10231 type strain and an echinocandin-resistant isolate (DPL20, FKS F645P). Caspofungin MICs, time-kill and postantifungal effect (PAFE) tests were performed in RPMI-1640 with and without 50 % serum. Killing rates (k values) in time-kill and PAFE experiments were determined for each strain and concentration. In time-kill experiments, colony count decreases were isolate- and concentration-dependent at 0.25, 1, 4, 8, 16 and 32 mg/L in RPMI-1640, but concentration-independent at 1, 4, 8, 16 and 32 mg/L in 50 % serum. One-hour caspofungin exposure at 4, 16 and 32 mg/L resulted in CFU decreases comparable with the results obtained in time-kill experiments in RPMI-1640, but 50 % serum at 4, 16 and 32 mg/L allowed growth of all isolates (k values were negative) (P < 0.05-0.001). PAFE in 50 % serum decreased markedly at 4, 16 and 32 mg/L. Killing rates remained high and concentration-independent in 50 % serum in case of continuous but not in case of brief caspofungin exposure. As only a short growth inhibition without killing was observed in 50 % serum, clinical relevance of caspofungin PAFE in vivo is questionable.

We compared the in vivo efficacy of single large dose of caspofungin to that of daily smaller caspofungin doses (with same cumulative doses) against C. albicans (echinocandin susceptible and resistant isolates) and the “psilosis� group in a neutropenic murine model. Seven treatment groups were formed for C. orthopsilosis, C. metapsilosis and C. albicans (no treatment, 1, 2 and 3 mg/kg caspofungin daily for five days; single 5, 10 and 15 mg/kg caspofungin doses). For C. parapsilosis there were five treatment groups (no treatment, 3 and 4 mg/kg caspofungin daily for five days; single 15 and 20 mg/kg caspofungin). Tissue burdens of C. orthopsilosis and C. parapsilosis were significantly decreased by daily 3 mg/kg and 10 or 15 mg/kg single caspofungin doses (P<0.05-0.01) and daily 4 mg/kg and by single 15 and 20 mg/kg caspofungin doses (P<0.05-0.01), respectively. Against C. metapsilosis all treatment arms except the daily 1 mg/kg were effective (P<0.05-<0.001). Against C. albicans all treatment doses were effective. Neither daily 16 mg/kg nor single 80 mg/kg were effective against the resistant C. albicans strain. Higher doses and less frequent administration of caspofungin were comparable or sometimes superior to the lower, daily-dose regimen against the “psilosis� group supporting further studies with this therapeutic strategy.

Caspofungin activity was determined in vitro and in vivo against three Candida orthopsilosis, three C. metapsilosis, two C. parapsilosis sensu stricto and two C. albicans isolates. MIC values and killing activity were determined in RPMI-1640 plus 50 % human serum. Neutropenic (cyclophosphamide-treated) mice were infected intravenously. Five-day intraperitoneal treatment with caspofungin was started after 24 h postinfection. Kidney burden was analyzed using the Kruskal-Wallis test with Dunn's post-test. In killing studies, caspofungin was fungistatic and fungicidal against C. albicans at ≥0.25 and ≥2 μg/ml concentrations, respectively. Caspofungin was fungistatic at ≥8-16, ≥2-8 and at ≥2-8 μg/ml against C. parapsilosis, C. orthopsilosis and C. metapsilosis, respectively. In the murine model, C. albicans was inhibited by 1, 2 and 5 mg/kg of caspofungin (P < 0.001 compared to the controls). Against C. parapsilosis, only 5 mg/kg caspofungin was effective against both isolates (P < 0.05). Two and five mg/kg of caspofungin was effective against all C. orthopsilosis and C. metapsilosis isolates (P < 0.05 to <0.001). Serum-based killing tests proved to be useful in predicting in vivo efficacy of caspofungin against four Candida species. Caspofungin at clinically attainable concentrations proved to be effective against all four species.

Background: The efficacy of fluconazole (FLU), amphotericin B (AMB) and caspofungin (CAS) was tested against three Candida orthopsilosis, three C. metapsilosis and two C. parapsilosis sensu stricto isolates in neutropenic mice., Methods: Mice were immunosuppressed by 200 mg/kg cyclophosphamide. Five-day intraperitoneal treatment was started 24 h after infection. Kidney burden was analyzed using the Kruskal-Wallis test., Results: FLU 10 and 20 mg/kg as well as AMB 1 mg/kg significantly decreased the fungal burden (p < 0.05) for all eight isolates of the three species. CAS 2 and 5 mg/kg were efficacious against all C. orthopsilosis and C. metapsilosis isolates (p < 0.05), but only 5 mg/kg CAS was effective against C. parapsilosis isolates (p < 0.05)., Conclusions: The efficacy of FLU and AMB against the three species was comparable. Though the activity of CAS was higher against C. orthopsilosis and C. metapsilosis, the current treatment guidelines for C. parapsilosis sensu stricto seem to be applicable to other 'psilosis' species., (Copyright © 2012 S. Karger AG, Basel.)

We compared the efficacy of single six mg/kg and 2x3 mg/kg caspofungin doses to the traditional one mg/kg daily against C. albicans in a neutropenic murine model. In lethality experiments, all regimens improved survival (p<0.0014 for all three isolates); differences among the treated groups were not statistically significant. We calculated kidney fungal burdens on each study day for six days postinfection using two isolates in two experiments. In the first three days, only the six mg/kg dose produced significant decrease on all study days (p<0.05-0.001), but differences between the three treatment arms disappeared by 4-6 days postinfection (p<0.05 for all isolates on all days)., (© E.S.I.F.T. srl - Firenze)

We investigated the activity of caspofungin against a Candida tropicalis clinical isolate showing paradoxical growth in vitro. BALB/c mice immunosuppressed by cyclophosphamide were infected intraperitoneally using 10(7) CFU/mouse. Caspofungin was administered intraperitoneally once daily for 5 days or as a single dose using the following doses: 0.12, 0.25, 1, 2, 3, 5, and 15 mg/kg. The single dose of caspofungin was effective only at 5 and 15 mg/kg concentrations (100% survival). Five-day caspofungin treatment led to 100% survival at doses of 1 mg/kg or higher. Caspofungin treatment significantly decreased the number of viable yeasts in the peritoneal lavage samples as well as in the infected abscesses at doses 1, 3, 5, and 15 mg/kg caspofungin as compared to the untreated control (P<0.001 in all cases), and even to the group treated with 0.12 mg/kg caspofungin (P<0.05 in all cases). At 2 mg/kg caspofungin dose, sterilization of the internal organs was reproducibly incomplete, suggesting that the role of paradoxical growth in the late clinical failure cannot be excluded.